Methods of isolating and detecting metal ions from proteins

ABSTRACT

A method of detecting a metal ion in a protein-containing sample includes adding a protein degrading enzyme to the protein-containing sample to form an enzyme degradation product, adding an acid to the enzyme degradation product to provide a mixture, filtering the mixture to provide a supernatant, extracting the supernatant with an organic solvent to remove organic solvent soluble byproducts to provide a washed aqueous layer, and detecting the metal ion in the washed aqueous layer. The method is amenable to the detection of heavy metal ions in complex products such as milk. A kit includes reagents for performing the method.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. patent application Ser.No. 15/255,756 filed Sep. 2, 2016, which is incorporated herein in itsentirety.

BACKGROUND

The present disclosure relates to metal ion isolation and detection.More particularly, the present disclosure relates to methods forisolation and detection of metal ions from proteins.

Metals (as part of compounds or as ions), including heavy metals, arepollutants gaining more attention due to potential toxicity which canhave lethal effects on living systems and general health. One source ofexposure to such metals is food, in particular dairy products such asmilk. Detection and monitoring of metals is highly desirable, in view ofcontinuing incidences of contaminated milk products in several parts ofthe world.

Existing technologies to detect metals, heavy metals in particular, areexpensive, time-consuming or use large (or bulky) devices that require aspecialized laboratory and make their use for field detectionimpractical.

Milk presents a typical example of interest for the metal detectionproblem, in foodstuff. Milk is an emulsion or colloid of butterfatglobules within an aqueous solution. The exact components of raw milkvaries but in general it contains significant amounts of lactose, fat,proteins and minerals as well as vitamins. The composition creates asignificant problem for field assay detection techniques because suchfield devices typically rely on biomolecule-based assays employing DNA,RNA or proteins. A complex matrix such as milk can create significantinterference and may require time-consuming sample preparation.

Thus, there is a need to provide improved solutions for isolation anddetection of metal ions from complex protein-containing substrates, suchas milk, that avoid the above-mentioned drawbacks. The presentdisclosure provides new methods to address these and related issues.

SUMMARY

In some aspects, embodiments herein relate to methods of detecting ametal ion in a protein-containing sample comprising adding a proteindegrading enzyme to the protein-containing sample to form an enzymedegradation product, adding an acid to the enzyme degradation product toprovide a mixture, filtering the mixture to provide a supernatant,extracting the supernatant with an organic solvent to remove organicsolvent soluble byproducts to provide a washed aqueous layer, anddetecting the metal ion in the washed aqueous layer.

In some aspects, embodiments herein relate to methods of detecting aheavy metal ion in a milk sample comprising adding a proteinase K enzymeto the milk sample to form an enzyme degradation product, adding nitricacid to the enzyme degradation product to provide a de-emulsifiedmixture, filtering the de-emulsified mixture to provide a supernatant,extracting the supernatant with chloroform to remove chloroform solublebyproducts to provide a washed aqueous layer, and detecting the heavymetal ion in the washed aqueous layer.

In some aspects, embodiments herein relate to kits comprising acontainer for holding a protein-containing sample, a proteinase Kreagent, a nitric acid reagent, a chloroform reagent, and instructionsfor performing the isolation of metal ions from the protein-containingsample.

BRIEF DESCRIPTION OF DRAWINGS

Various embodiments of the present disclosure will be described hereinbelow with reference to the figures wherein:

FIG. 1 shows a method for the isolation and detection of metal ions froma protein-containing sample, in accordance with embodiments herein.

FIG. 2 shows a plot indicating the effectiveness of the acid onextraction of lead ion from milk via fluorescence detection.

FIG. 3 shows a plot indicating the effectiveness of the organic solventon extraction of lead ion from milk via fluorescence detection.

FIG. 4 shows a plot indicating the effectiveness of the type of membraneon extraction of lead ion from milk via fluorescence detection.

FIG. 5 shows another plot indicating the effectiveness of the type ofmembrane on extraction of lead ion from milk via fluorescence detection.

FIG. 6 shows a plot indicating the effectiveness of proteinase K versusa control with no enzyme digestion, on heavy metal extraction from milkvia fluorescence detection.

FIG. 7A. shows a plot of lead (II) concentration (nM) versusfluorescence (a.u.) from samples isolated from milk indicating theability to quantitatively detect lead.

FIG. 7B shows a plot of uranium (VI) concentration (nM) versusfluorescence (a.u.) from samples isolated from milk indicating theability to quantitatively detect uranium.

FIG. 8A shows a plot of lead (II) concentration (nM) versus GMR signal(a.u.) from samples isolated from milk indicating the ability toquantitatively detect lead.

FIG. 8B shows a plot of uranium (VI) concentration (nM) versusfluorescence (a.u.) from samples isolated from milk indicating theability to quantitatively detect uranium.

DETAILED DESCRIPTION

Embodiments herein provide processes for isolating metals, includingheavy metals from protein-rich substrates, such as from milk. In anexemplary method isolating heavy metals from milk in the Examples below,three facile steps are used to remove interfering proteins and lipids.In some such embodiments, the first step may employ an enzyme to degradethe proteins in the milk. In the second step, acid is added tode-emulsify the milk. In the third step, a nitrocellulose filter is usedto bind the remaining proteins, lactose and fat. These three steps maybe generally used in a methodology to facilitate detection of metal ionsfrom protein sources and foodstuffs, more generally. This process issimple, avoids use of any specialized laboratory instrument and is rapidwith a timescale for detection in a range from about 45 to about 60minutes total. Additional reagents, such as anionic surfactants, may beemployed to accelerate the detection time by increasing the rate of theenzyme degradation step in the processes disclosed herein.

The methods herein are general and are exemplified herein in thedetection of two different heavy metal ions (lead and uranium) in milk.The isolated metal ions can be quantitatively detected using anybioassay. The bioassays for the actual detection step are not limited.For example, one can use traditional fluorescence detection methods or agiant magnetoresistance (GMR) platform, as disclosed in pending U.S.Application Publication No. 2016/0011182, which is incorporated hereinby reference in its entirety. Other detection methodss includecolorimetric methods, such as with horseradish peroxidase,chemiluminescence, or electrochemical methods.

In some embodiments, there are provided methods of detecting a metal ionin a protein-containing sample comprising adding a protein degradingenzyme to the protein-containing sample to form an enzyme degradationproduct, adding an acid to the enzyme degradation product to provide amixture, filtering the mixture to provide a supernatant, extracting thesupernatant with an organic solvent to remove organic solvent solublebyproducts to provide a washed aqueous layer, and detecting the metalion in the washed aqueous layer.

In some embodiments, any one of the aforementioned steps in the methodmay be omitted depending on the nature of the protein-containing sample.For example, in some embodiments, it may be possible to omit adding theacid to the enzyme degradation product. While this step is useful in theexample of milk as an aid to de-emulsify the milk, other samples may notrequire such treatment because they may not be emulsions to begin withor the protein-degrading enzyme may be sufficient to break down theprotein-containing sample to cause de-emulsification on its own. Inother embodiments, the protein degrading enzyme may be omitted. In suchembodiments, treatment of a protein-containing sample with strong acids,such as concentration nitric acid, may be sufficient. Thus, in someembodiments, there are provided methods that do not employ a proteindegrading enzyme. In some such embodiments, the methods do not employ aproteinase K. In other embodiments, the methods do not employ a strongacid.

In some embodiments, the protein-containing sample is liquid milk. Inother embodiments, the protein-containing sample is powdered milk. Instill further embodiments, the protein-containing sample is a solutionof a protein powder. Thus, methods herein can be used to detect metals,including heavy metals, in a variety of consumer products, including butnot limited to protein supplements and related nutraceuticals. In someembodiments, methods disclosed herein are particularly suited to thedetection of metals in dairy products including, without limitation,butter, milk, cheese, crème, yogurt, sour cream, whey products,evaporated milk, buttermilk, infant formula products, milk proteinconcentrates, milk hydrolysates, and caseinates. The protein-containingsample is not limited to dairy-based products, with the methods beingamenable to metal detection in plant and nut-based milks as well,including milks derived from soy, almonds, hazelnuts, cashews, and thelike.

In some embodiments, the protein degrading enzyme may be a proteinase Kenzyme. Other protein degrading enzymes may be used alone or incombination with proteinase K including, without limitation, pserineproteases, threonine proteases, cysteine proteases, aspartic proteases,and glutamic proteases. Other protein degrading enzymes may include,without limitation, digestion enzymes such as pepsin, trypsin,chymotrypsin, metalloprotease, and elastase.

In embodiments, methods may further include the use of a denaturingagent. The denaturing agent may be particularly beneficial added duringthe initial protein degrading step with the protein degrading enzyme.Without being bound by theory, the denaturing agent may provide a moreopen protein structure facilitating access by the protein degradingenzymes. In such embodiments, the denaturing agent may be an anionic ornon-ionic surfactant, urea, a chelating agent, a sulfhydryl reagent, aserine protease, or combinations thereof.

Anionic or non-ionic surfactants may include one or more of ammoniumlauryl sulfate, potassium lauryl sulfate, sodium alkyl sulfate, sodiumdodecyl sulfate, sodium dodecylbenzenesulfonate, sodium laurate, sodiumlaureth sulfate, sodium lauroyl sarcosinate, sodium myreth sulfate,sodium stearate, and the similar anionic surfactants. Non-ionicsurfactants may include Tween-20.

Chelating agents may include any multifunctional organic capable ofcoordinating to a metal ion. Suitable examples include, withoutlimitation, EDTA (ethylenediaminetetraacetic acid), EGTA (ethyleneglycol tetraacetic acid), polyethylene glycol, propylene glycol,citrates, gluconates, alkylenepolyamine polyacetic acid and saltthereof, salt of alkylenepolyamine polyacetic acid, nitrilotriaceticacid (NTA), thiosulfate, iminodiacetic acid, and alkylenepolyaminopolycarboxylic acids. In some embodiments, the chelating agent may beselected to be specific for the query metal ion in the method. In someembodiments, the chelating agent may be selected to remove non-querymetal ions.

In some embodiments, the methods may include a second step that is astrong acid treatment step. In some such embodiments, the acid may beconcentrated nitric acid. In other embodiments, the acid may behydrochloric acid or an acid buffer such as sodium acetate. Theconcentration of the acids may be about 1M whereas the acid buffers maybe about 3M.

In some embodiments, the filtering step is performed with about a 0.2micron pore size filter, although a range of sizes from about 0.05microns to about 1 micron may be useful. In some embodiments, the filtermay be a nitrocellulose filter.

In embodiments, the detecting step is performed by fluorescencedetection. In other embodiments, the detecting step is performed bygiant magnetoresistance (GMR) measurements. In some embodiments, thedetecting step may be performed by colorimetric methods or withelectrochemical sensors. The Examples below show the use of thesedetection methods in practice.

U.S. Application Publication No. 2016/0011182 describes the use of GMRin a magnetic sensor having one or more layers formed on a base forsensing a magnetic field created by magnetic particles present inproximity to the magnetic sensor. A first end of each of a first set ofstrands (designed DNA or RNA that is query metal ion selective) isimmobilized with respect to the magnetic sensor. A magnetic particle isattached to a second end of each of the first set of strands so thatwhen a sample containing a query metal ion is in contact with the base,the query metal ion causes at least some of the first set of strands tobreak resulting in the magnetic particle attached to the second end ofeach of the at least some of the first set of strands to no longer be inproximity to the magnetic sensor. The change can be measured with anappropriate interface of the detection device. In some embodiments,other conventional GMR detection motifs may be used.

In some embodiments, there are provided methods of detecting a heavymetal ion in a milk sample comprising adding a proteinase K enzyme tothe milk sample to form an enzyme degradation product adding nitric acidto the enzyme degradation product to provide a de-emulsified mixturefiltering the de-emulsified mixture to provide a supernatant, extractingthe supernatant with chloroform to remove chloroform soluble byproductsto provide a washed aqueous layer, and detecting the heavy metal ion inthe washed aqueous layer.

Referring now to FIG. 1, there is shown a schematic diagram of anexemplary method 100 for detecting heavy metal ions in a milk sample. instep 10 the sample is incubated with a proteinase K enzyme to degradethe proteins. Nitric acid (concentrated) may be added at step 20. Thismay serve to both de-emulsify and further denature the degraded enzymefrom step 10. At step 30, the sample is then passed through a filter,such as a nitrocellulose filter. Chloroform or other suitable organicsolvent for bilayer extraction is then added and thoroughly mixed atstep 40. At step 50 the aqueous layer containing the query heavy metalion is separate from the organic layer. Finally, at step 60 the aqueouslayer is tested to detect the query heavy metal ion. This last step canbe performed in any manner enumerated herein, including by fluorescencedetection or GMR measurements.

In some embodiments, the heavy metal ion is lead. In some embodiments,the heavy metal ion is uranium. In some embodiments, the methods fordetecting heavy metal ions in milk are designed to detect withselectivity for particular heavy metal ions. In some embodiments, thedetecting methods may be designed to detect two or more heavy metal ionssimultaneously, such as two, three, four, or even five heavy metal ions.

In some embodiments, there are provided kits comprising a container forholding a protein-containing sample, a proteinase K reagent, a nitricacid reagent, a chloroform reagent and instructions for performing theisolation of metal ions from the protein-containing sample. In someembodiments, the kit is a small scale kit for performing the isolationof metal ions in the field. In some embodiments, kits may furthercomprise a hand held detection device for detecting metal ions. In somesuch embodiments, the hand held detection device may employ fluorescencedetection or detection based on giant magnetoresistance (GMR).

The kits may further comprise other protein denaturing agents, includingat least one selected from the group consisting of sodium dodecylsulfate (SDS), urea, ethylenediamine tetraacetic acid, trypsin, andchymotrypsin. Kits may also contain the requisite buffers, vials ofdeionized water, and other reagents.

The following Examples are being submitted to illustrate embodiments ofthe present disclosure. These Examples are intended to be illustrativeonly and are not intended to limit the scope of the present disclosure.Also, parts and percentages are by weight unless otherwise indicated. Asused herein, “room temperature” refers to a temperature of from about20° C. to about 25° C.

EXAMPLES Example 1

This example describes an exemplary method for isolating heavy metalsfrom milk and detection by fluorescence and with a magnetic sensor.

Digestion of Proteins

The main biomolecular components in milk and milk-based products areproteins and lipids. They are also responsible for interference withmost bioassays. To remove proteins, this Example employs a proteindigesting enzyme. Although there are many such protein digesting enzymesthat are potentially useful, this Example uses Proteinase K. ProteinaseK is active under a diverse set of conditions, works at room temperatureand does not need any special buffer. It is also a common enzyme inbiochemistry, especially where removal of proteins are required withoutaltering DNA or RNA (e.g., genomic DNA extraction from bacterial cells).Proteinase K digestion time can vary. The minimum time required may beabout 15 minutes in the presence of about 72 units of proteinase K inmilk at room temperature.

Choice of Acid

To optimize the process in milk, several acids to de-emulsify the milkwere tested, including nitric acid (1M), hydrochloric acid (1M) and alow pH sodium acetate buffer (3M, pH 5.2). The volume used for alltrials was 167.5 microliters. The results indicated that presence ofPb²⁺ ion can be detected with both acids and sodium acetate buffer. FIG.2. shows the effect of acid on extraction of lead ion from milk. Closedcircles indicate DNAzyme-based sensor's fluorescence signal in theabsence of lead and open circles indicate fluorescence signal obtainedin the presence of 500 nM Pb²⁺. As a control, DNAzyme-based sensor wasused to detect lead ion in water where no extraction from milk wasinvolved. This result provided a control and was used to assess theeffectiveness of the extraction protocol. When compared to just water,which acted as a control, nitric acid showed the best result. Therefore,for further experiments, nitric acid was acid employed. It is expectedthat sulfuric acid would work in this step just as well.

Choice of Organic Solvent for Extraction

Even after the milk is de-emulsified and passed through a nitrocellulosefilter, it was expected that there would be residual proteins and lipidsin the filtrate. In biochemistry, to remove trace amount of proteinsfrom nucleic acid solution, organic solvents are often used. A classicexample is phenol chloroform extraction of DNA or RNA after enzymaticprocess. In this process, the proteins denature in organic solvents andsettle in the interface of aqueous and organic layers. The salts andnucleic acids remain in aqueous layer. Since lipids are soluble innon-polar organic solvents, it was hypothesized that an organic solventextraction will also remove the lipids. Organic solvents that can beemployed include, without limitation, chloroform, ethyl acetate, and amixture of phenol-chloroform-isoamyl alcohol.

FIG. 3 shows the effect of organic solvent on extraction of lead ionfrom milk. Closed circles indicate fluorescence signal obtained in theabsence of lead, whereas open circles indicate fluorescence signalobtained in the presence of 500 nM Pb²⁺. Phe-CHCl3-IAA isphenol-chloroform isoamyl alcohol mixture; CHCl₃ is chloroform. As notedin FIG. 2, the result in water was used as a control and to assess theeffectiveness of the extraction protocol.

For phenol-chloroform-isoamyl alcohol, phase separation requiredcentrifugation. Additionally, the result showed sub-optimal activityafter extraction. For ethyl acetate and chloroform, phase separation canbe achieved without centrifugation. Both chloroform and ethyl acetateshowed the expected level of activity. However, chloroform wasdetermined to be the most user-friendly as the aqueous layer is the toplayer and thus easy to remove from the organic layer. For ethyl acetate,the aqueous layer is the bottom layer and thus its removal may be trickyfor a non-expert person.

Choice of Filter

Filtration was used to remove proteins and lipids and thus the choice ofmembrane was limited to polyvinylidene difluoride (PVDF) andnitrocellulose. Both membranes have high protein binding capacity andcan be obtained in several pore sizes. Nitrocellulose is suitable forbinding low molecular weight proteins. Since, proteinase K digested mostof the proteins into smaller fragments, efficient binding of lowmolecular weight protein was deemed ideal in this case. Among variouspore sizes available, 0.2 μm pore size was selected primarily to preventsmall peptide fragments to flow through while not clogging the filter.Regenerated nitrocellulose (0.2 μm pore size) was also used; however, noheavy metal ion was detected after sample preparation. This is believedto be primarily due to low protein binding capacity of regeneratednitrocellulose. FIG. 4 shows the effect of type of membrane onextraction. Filtration using regenerated nitrocellulose failed to detectPb²⁺ ion present in the milk.

As an alternative to nitrocellulose, PMMA and silica beads were alsoused. These beads are known to adsorb proteins on their surface.However, the signal obtained after extraction via beads indicated thatthey failed to efficiently remove proteins and lipids. FIG. 5 shows theeffect of filtration method on extraction of lead ion from milk. Closedcircles indicate fluorescence signal obtained in the absence of lead,whereas open circles indicate fluorescence signal obtained in thepresence of 500 nM Pb²⁺. As noted in previous Figures, the result inwater was used as a control and to assess the effectiveness of themethod.

Additional Combination of Process

Further attempts to simplify the process were made by systematicallyremoving one step at a time. This also allowed assessment of whethereach step was absolutely necessary to the overall success.

First, proteinase K was removed from the process and the processcommenced by adding 167.5 μL of nitric acid (1M) to the diluted milksample. After filtration through nitrocellulose and an extraction fromchloroform, the assay could detect the presence of Pb²⁺ ion in thesample. However, when compared against the control, it was found thatthe activity was not lower than expected. FIG. 6 shows the effect ofProteinase K on heavy metal extraction from milk.

Removal of the nitrocellulose filtration from the process was alsoassessed to determine whether a simple organic extraction is sufficientto remove proteins and lipids. Chloroform was used as the organicsolvent and during extraction, a large amount of proteins remain in theinterface. Thus, efficient extraction of aqueous layer became an issueand for complete removal, at least five consecutive extractions wererequired before the aqueous layer could be safely removed withoutdisturbing the interface. This creates complexity and additionally, itwas found that the activity of the sensor was sub-optimal. Thisindicates that the organic solvent extraction without nitrocellulosefiltration was not sufficient to remove proteins and lipids.

Process is Independent of Detection Method

Once heavy metal is extracted from the milk, it was detected by aDNAzyme-based sensors that are specific to a single metal ion. Thedetection method for the heavy metals can utilize either fluorescentdyes (Cy3, Cy5, FAM, etc.) or via a GMR method (i.e., magneticnanoparticles). All the results shown above utilized fluorescent dyeCy3. However, a similar result can be obtained via GMR as shown below.

Overview of the Heavy Metal Extraction Process

In a glass or plastic tube, 1 mL of whole milk was diluted to a finalvolume of 5 mL with deionized water. 90 μL of proteinase K (0.8 U/pL,New England Biolabs, Ipswich, Mass.) was added to the milk and incubatedat room temperature for 15 minutes. After 15 minutes of incubation,167.5 μL of 1M nitric acid was added to the tube and mixed well tode-emulsify the milk. A 1 mL aliquot of this de-emulsified milk samplewas removed using a 1 mL syringe and passed through a nitrocellulosefilter (0.2 μm pore size, Maine Engineering). Success of the downstreambioassay depends on the efficient filtration and thus, any whitesuspension in the filtrate indicates that nitrocellulose filter failedto efficiently remove the proteins and lipids. In such a case, theprocess was repeated again. The clear filtrate was collected in a 1.5 mLcentrifuge tube and the total volume of the filtrate ranged from 200-400μL, enough for downstream DNAzyme-based assays.

Following filtration, an equal volume of chloroform was added to thefiltrate, mixed well and then the tube was allowed to stand for 5 min.The top aqueous layer was carefully removed using a pipette and placedin a new 0.5 mL centrifuge tube. 50 μL of this sample was used forassays to determine the presence of metal ions using functionalDNAzyme-based assays as described below. FIGS. 7A and 7B show thequantitative determination of Pb²⁺ and UO₂ ²⁺ after extraction frommilk. The data in the absence of heavy metal ions were obtained by notspiking milk with any heavy metal ion solution.

DNAzyme-Based Assay to Detect Heavy Metals

For assays, DNA microarrays were constructed by immobilizing substrateDNA on a glass slide containing Codelink™ surface (Surmodics, EdenPrairie, Minn.). The substrates were dissolved in a printing buffercontaining sodium phosphate and polyvinyl alcohol and arrayedrobotically onto glass slides with a distance of 400 μm between thecenters of adjacent spots using a piezoelectric spotting robot (ScienionAG, Berlin, Germany). Printing was performed in an enclosed cabinet atabout 18° C. and about 70% humidity.

After printing, the microarrays were kept in a humid chamber at roomtemperature for about 12 to 16 hours. The substrates were immobilized onglass slides via a reaction between NHS ester group on the surface and3′-terminal primary amine on the substrates. The excess functionalgroups on the slide were blocked using a solution of 50 mM ethanolamine.Following the blocking with ethanolamine, the slides were washed withde-ionized water and spin-dried. The microarrays were generated so thatthere were twelve sub-arrays per glass slide and sub-arrays wereseparated by placing a polystyrene ring around each sub-array. Thisprocess created twelve reaction chambers per slide.

DNAzyme sensors, 1000-fold in excess over immobilized substrate, weredissolved in reaction buffer, 85 μL of the solution was added to thereaction chamber and the reaction was initiated by adding 0.85 μL ofheavy metal ion solution. The reaction was allowed to proceed for 15minutes and then the solution was removed from the reaction chamber viapipette. The reaction chamber was washed twice with 85 μL of reactionbuffer to remove residual cleaved substrate and excess DNAzyme sensor.

To detect using fluorescence, to the reaction chamber 90 μL ofCy3-streptavidin dye (5 μg/mL in reaction buffer) was added andincubated at room temperature for 30 minutes. Following removal of thedye, the slides were washed with 0.5% sodium dodecyl sulfate solutionand de-ionized water. The slides were imaged on the Axon GenePix 4000B(Axon Instruments, Foster City, Calif.) scanner with 5 μm resolutionusing a Cy5/Cy3 optical filter. The laser power and photomultiplier tubevoltage (PMT) were set to gain optimum signal intensities. The original16-bit tiff images were quantified with GenePix software 6.0 (AxonInstruments, Foster City, Calif.). To detect using GMR, to the reactionchamber 80 μL of streptavidin coated magnetic nanoparticle coated wasadded and signal detected and recorded using a GMR device. FIGS. 8A and8B show the detection of lead and uranium using the GMR sensors.

What is claimed:
 1. A method of detecting a metal ion in aprotein-containing sample comprising: adding a proteinase K enzyme tothe protein-containing sample to form an enzyme degradation product;adding concentrated nitric acid to the enzyme degradation product toprovide a mixture; filtering the mixture to provide a supernatant;extracting the supernatant with an organic solvent to remove organicsolvent soluble byproducts to provide a washed aqueous layer; anddetecting the metal ion in the washed aqueous layer in a handheld GMRsensing device.
 2. The method of claim 1, wherein the protein-containingsample is liquid milk.
 3. The method of claim 1, wherein theprotein-containing sample is powdered milk.
 4. The method of claim 1,wherein the protein-containing sample is a solution of a protein powder.5. The method of claim 1, further comprising a denaturing agent.
 6. Themethod of claim 1, wherein the denaturing agent is an anionicsurfactant, a urea, a chelating agents, a sulfhydryl reagent, a serineprotease, or combinations thereof.
 7. The method of claim 1, wherein thefiltering step is performed with a 3 micron to 5 micron pore sizefilter.
 8. A method of detecting a heavy metal ion in a milk samplecomprising: adding a proteinase K enzyme to the milk sample to form anenzyme degradation product; adding nitric acid to the enzyme degradationproduct to provide a de-emulsified mixture; filtering the de-emulsifiedmixture to provide a supernatant; extracting the supernatant withchloroform to remove chloroform soluble byproducts to provide a washedaqueous layer; and detecting the heavy metal ion in the washed aqueouslayer in a handheld GMR sensing device.
 9. The method of claim 8,wherein the heavy metal ion is lead. The method of claim 8, wherein theheavy metal ion is uranium.